Download White matter tract alterations in fragile X

American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 118B:81 – 88 (2003)
White Matter Tract Alterations in Fragile
X Syndrome: Preliminary Evidence From
Diffusion Tensor Imaging
Naama Barnea-Goraly,1 Stephan Eliez,1 Maj Hedeus,2 Vinod Menon,1 Christopher D. White,1
Michael Moseley,2 and Allan L. Reiss1*
1
Department of Psychiatry & Behavioral Sciences, Stanford University School of Medicine, Stanford, California
Department of Radiology, Stanford University School of Medicine, Stanford, California
2
Fragile X syndrome, the most common form
of hereditary mental retardation, causes
disruption in the development of dendrites
and synapses, the targets for axonal growth
in the central nervous system. This disruption could potentially affect the development, wiring, and targeting of axons. The
current study utilized diffusion tensor imaging (DTI) to investigate whether white
matter tract integrity and connectivity are
altered in fragile X syndrome. Ten females
with a diagnosis of fragile X syndrome and
ten, age matched, female control subjects
underwent diffusion weighted MRI scans. A
whole brain analysis of fractional anisotropy
(FA) values was performed using statistical
parametric mapping (SPM). A follow-up,
regions-of-interest analysis also was conducted. Relative to controls, females with
fragile X exhibited lower FA values in white
matter in fronto-striatal pathways, as well as
in parietal sensory-motor tracts. This preliminary study suggests that regionally specific alterations of white matter integrity
occur in females with fragile X. Aberrant
white matter connectivity in these regions is
consistent with the profile of cognitive and
behavioral features of fragile X syndrome,
Grant sponsor: NIH; Grant numbers: MH01142, HD31715,
MH50047; Grant sponsor: The Packard Foundation; Grant
sponsor: The Sinclair Fund; Grant sponsor: The Lynda and Scott
Canel Fund for Fragile X Research.
*Correspondence to: Allan L. Reiss, M.D., 401 Quarry Road,
Department of Psychiatry & Behavioral Sciences, Stanford
University School of Medicine, Stanford, CA 94305-5719.
E-mail: [email protected]
Received 17 May 2002; Accepted 4 September 2002
DOI 10.1002/ajmg.b.10035
ß 2003 Wiley-Liss, Inc.
and potentially provide additional insight
into the detrimental effects of suboptimal
levels of FMRP in the developing brain.
ß 2003 Wiley-Liss, Inc.
KEY WORDS: brain; DTI; MRI
INTRODUCTION
Fragile X syndrome has been recognized widely as a
distinct clinical syndrome only since the 1970s. However, it is now known to be the most frequent inherited
form of neurodevelopmental disability in humans, with
an estimated incidence of one in 3,000–4,000 [Kemper
et al., 1988; Crowe and Hay, 1990; Grigsby et al., 1990;
Freund, 1991; Freund et al., 1993; Mazzocco et al., 1993;
Baumgardner et al., 1994]. Fragile X syndrome arises
from a single gene mutation on the X chromosome,
causing reduced levels of the protein, fragile X mental
retardation protein (FMRP), a product of the fragile X
mental retardation 1 gene (FMR1).
Low or absent levels of FMRP affect physical appearance, and increase risk for psychiatric, cognitive, and
behavioral disability. Physical manifestations of fragile
X syndrome include a long and narrow face, large dysmorphic ears, a prominent jaw and, in males, macroorchidism [Loesch et al., 1988; Davids et al., 1990;
Meryash et al., 1984]. Males are usually more affected
than females and have moderate to severe mental retardation, whereas females typically have mild mental
retardation or normal intellectual functioning accompanied by learning disabilities [Riddle et al., 1998]. The
cognitive phenotype of fragile X syndrome includes
increased risk for deficits in working memory, short
term memory, visuospatial abilities, visual-motor coordination, arithmetic reasoning, and executive function [Kemper et al., 1988; Crowe and Hay, 1990; Grigsby
et al., 1990; Freund, 1991; Freund et al., 1993; Mazzocco
et al., 1993; Baumgardner et al., 1994]. Behaviorally,
individuals with fragile X are at risk for exhibiting
hyperactivity, abnormal social communication, language impairments, unusual response to sensory stimuli, gaze avoidance, anxiety, and stereotypic behavior
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Barnea-Goraly et al.
[Simon, 1990; Reiss and Freund, 1992; Lachiewicz and
Dawson, 1994; Baumgardner et al., 1995; Turk and
Cornish, 1998]. This recognizable cognitive, and behavioral profile associated with fragile X implies extensive
involvement of FMRP in multiple brain functions and
suggests that the study of individuals with this condition may provide insights into the pathophysiology of
pediatric neuropsychiatric and learning disorders that
share similar deficits.
FMR1 mRNA expression within the brain is limited to
neurons, and is highly expressed throughout the fetal
and adult human brain [Devys et al., 1993; Hinds et al.,
1993; Feng et al., 1997; Tamanini et al., 1997; Agulhon
et al., 1999]. Within neurons, FMRP is found primarily
in the perikaryon, in dendrites of all calibers, and in
synapses [Devys et al., 1993; Feng et al., 1997; Weiler
et al., 1997]. Early anatomical examination of the fragile
X brain found no abnormalities in gross neuropathological examinations [Rudelli et al., 1985; Hinton et al.,
1991]. Histologic examinations, however, detected abnormalities in the dendritic structure and density, as
well as immature synapses in subjects with fragile X
syndrome. These were also observed in FMR1 knockout
mice [Rudelli et al., 1985; Hinton et al., 1991; Comery
et al., 1997; Irwin et al., 2001]. Neuroimaging studies of
fragile X syndrome indicate that the FMR1 full mutation has both a general effect on brain development and
a selective effect on the growth and maintenance of
specific brain regions. Specifically, studies have described increased cerebral and ventricular volumes [Reiss
et al., 1991; Wisniewski et al., 1991; Schapiro et al.,
1995], as well as enlarged caudate nucleus [Reiss et al.,
1995a,b] and hippocampal volumes [Reiss et al., 1994;
Kates et al., 1997], and decreased cerebellar vermis area
[Reiss et al., 1991; Mostofsky et al., 1998].
Although FMRP is primarily expressed in the cell
body and postsynaptic regions, the observed dysmorphology of dendrites and synapses in fragile X syndrome
could also potentially affect the development, wiring,
and targeting of axons that link affected brain regions.
This, in turn, could influence white matter density and
coherence between these areas. To investigate the structure of white matter tracts in fragile X syndrome we
used diffusion tensor imaging (DTI), a recently developed magnetic resonance (MR) imaging technique that
enables the investigation of the orientation of brain
pathways in vivo. In axons, water diffusion is impeded
by cell walls and myelin sheaths. As a result, water
movement along the axis of an axon is much larger than
water movement perpendicular to it. DTI allows visualization of this movement by fully characterizing water
diffusion in three-dimensional space [Basser et al.,
1994]. Since the movement of water molecules is
restricted by the boundaries of the axons, visualization
of that movement allows visualization of the structure
and direction of axons within a DTI brain image.
This study investigated white matter structure in
individuals with fragile X syndrome. Based on previous
neuroimaging research involving individuals with fragile X that indicated dysfunction of frontal-subcortical
circuits [Hjalgrim et al., 1999], and the finding of an
enlarged caudate nucleus in fragile X [Reiss et al.,
1995a,b], we hypothesized that there would be white
matter differences between subjects with fragile X and
controls in pathways connecting the corpus striatum
and the frontal lobe.
METHODS
Subjects
Ten female subjects with a diagnosis of fragile X syndrome (age range ¼ 13.1–22.7 years; mean: 16.69 3.82) and ten, age matched, healthy females (age
range ¼ 11.5–22.6 years; mean 17.05 3.82) participated in the current study. Subjects with fragile X were
recruited through advertisement in newsletters. Control subjects were recruited through ads in newspapers
and parent networks. All control subjects were in good
health and without evidence of neurological or psychiatric disorder. Control subjects with IQ of above 130 were
excluded from the study as they may not represent the
average population. Females were chosen because of
their higher likelihood to complete a MRI study without
need of sedation [Reiss et al., 1995a,b]. After providing a
complete description of the study to all participating
subjects and their caretakers, written informed consent
was obtained under protocols approved by the Institutional Review Board of Stanford University.
The diagnosis of fragile X syndrome was confirmed by
the presence of an FMR1 full mutation through DNA
analysis. Standard Southern blot and polymerase chain
reaction analyses were performed followed by FMR1specific probe hybridization [Oberele and Rousseau,
1991]. The CGG repeat number was calculated from
the Southern blot autoradiogram images. Additionally,
each participant underwent cognitive (IQ) testing using
the Wechsler Intelligence Scale for Children (WISC-III)
for subjects under 17 years of age, and the Wechsler
Adult Intelligence Scale (WAIS-III) for subjects aged
17 and above.
Image Acquisition
Each participant was scanned on a 1.5 T whole body
GE Signa Horizon scanner (GE medical systems,
Milwaukee). A DTI sequence was based on a single-shot
spin-echo echo-planar imaging (EPI) sequence with
diffusion sensitizing gradients applied on either side of
the 1808 refocusing pulse [Moseley et al., 1991; Basser
et al., 1994]. Imaging parameters for the diffusion
weighted sequence were as follows: field of view (FOV) ¼
24 cm, matrix size 128 128, TE/TR ¼ 106/6,000 ms,
18 or 19 axial-oblique slices, slice thickness 5 mm/skip
1 mm. Seven scans in each group were zero filled to
256 256 matrix size due to database requirements at
the time of acquisition. This after-the-scan manipulation did not change the resolution or the signal to noise
ratio (SNR). The scan was prescribed from the top of the
brain and included only the most superior part of the
cerebellum. Diffusion gradient duration was d ¼ 32 ms,
diffusion weighting was b ¼ 900 s/mm2. In addition, T2
weighted image were acquired by removing the diffusion
sensitizing gradients.
White Matter Tract Alterations in Fragile X Syndrome
Diffusion was measured along six non-collinear
directions: XY, XZ, YZ, XY, XZ, and YZ. This pattern was repeated four times for each slice with the sign
of all diffusion gradients inverted for odd repetitions.
Image Processing
Raters blinded to diagnosis manually inspected the
raw data images for motion artifacts and corrupted
images were discarded. A maximum of two images
were discarded for each direction. Eddy current effects
in the diffusion weighted images (i.e., geometric distortions that vary from one diffusion direction to the next)
were unwarped prior to averaging, [de Crespigny and
Moseley, 1998]. Averaging of the four magnitude images
efficiently removed the effect of gradient cross-terms
between the diffusion sensitizing and imaging gradients
[Neeman et al., 1991]. For each slice, two T2 images with
no diffusion weighting (b ¼ 0 s/mm2) were acquired and
averaged.
In the current study, fractional anisotropy (FA) was
the variable of interest. FA is an intravoxel measure that
yields values between 0 (perfectly isotropic diffusion)
and 1 (perfectly anisotropic diffusion) [Basser and
Pierpaoli, 1996; Pierpaoli and Basser, 1996]. The degree
of diffusion anisotropy in a voxel is determined by
microstructural features of the tissue in that particular
image voxel, including fiber diameter and density, as
well as macrostructural features such as intravoxel
fiber-tract coherence, and also by the degree of myelination [Pierpaoli and Basser, 1996]. The greater the diffusion anisotropy within a measured voxel—the higher
the FA value. In fragile X syndrome, we expected to find
reduced FA values compared to control subjects due to
reduced fiber density or coherence.
The FA was calculated for each voxel according to
Basser and Pierpaoli [1996] to produce an FA image. The
FA images were further processed using Statistic Parametric Mapping software (SPM99 software, Wellcome,
UK). The T2 weighted image map was used to determine
normalizing parameters subsequently applied to the FA
images using SPM99. Re-sampling in the normalization
process eliminates potential differences due to different
matrix sizes. Normalized FA images were smoothed
with a 4 mm kernel in order to increase the SNR. These
smoothed images for controls and subjects with fragile X
were compared using voxel-wise two-tailed t-test statistics; t-scores were normalized to Z scores to provide a
statistical measure of group differences that are independent of sample size. The resultant Z score reflects
the voxel-wise difference in FA between the two groups.
Finally, in order to determine the presence of significant clusters of differences, the joint expected probability distribution of the height and extent of Z scores
with height (Z > 2.33; P < 0.01) and extent (Z > 1.67;
P < 0.05) thresholds, was used in order to correct for
spatial correlation in the data [Poline et al., 1997].
Using a normalized, average SPGR image for subjects
with fragile X and control subjects, a white matter
cerebral mask was created and used to highlight changes in white matter tracts eliminating noise and edge
effects.
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A confirmatory analysis was subsequently conducted
using regions of interest (ROIs). Spherical ROIs with a
diameter of 2 mm were placed on the individual FA maps
in pathways that were delineated as having lower FA
values in our fragile X sample in the whole brain, voxelbased (SPM) analysis. This included the left frontalcaudate, and bilateral sensory-motor tracts. Although
not reaching statistical significance in the voxel-based
analysis, an ROI also was placed in the corresponding
frontal-caudate tract on the right side of the brain to
investigate possible laterality effects observed in fragile
X subjects. Finally, ‘‘control’’ ROIs were placed bilaterally in the occipital optic radiations. A nonparametric
test (Mann–Whitney U ) was used to examine group
differences. A P-value of 0.01 (two-tailed) was chosen as
the significance threshold.
RESULTS
Subjects with fragile X showed reduced FA in several
brain regions compared to controls. As shown in Figure 1,
one cluster was observed in left frontal-caudate white
matter tracts, extending between the head of the caudate
towards the prefrontal cortex. In addition, two welldefined clusters were observed along the corona radiata
and centrum semiovale corresponding to sensory-motor
areas bilaterally. A small cluster of increased FA in
subjects with fragile X when compared with controls was
seen in the posterior part of the superior temporal gyrus.
Examination of group differences by ROI confirmed
the significant FA differences observed in the voxelbased (SPM) analysis (Table I and Fig. 2). Significant
differences in FA values between groups were seen in
left frontal-caudate (U ¼ 6; P ¼ 0.009), left sensorymotor (U ¼ 5.5; P ¼ 0.0008), and in right sensory-motor
tracts (U ¼ 3; P ¼ 0.0004). Although a significant cluster
was not detected in the voxel-based analysis, the average FA value in the right frontal-caudate tract also was
found to be significantly lower in the fragile X group
(U ¼ 12.5; P ¼ 0.004). Average values generated from the
ROI placed in left and right optic radiations did not differ
between the two groups (left optic radiations: U ¼ 60;
P ¼ 0.4; right optic radiations: U ¼ 46.5; P ¼ 0.79).
IQ scores in the fragile X group ranged between 65 and
107 (mean 80.5 13.4), and in the control group between
99 and 128 (mean 115 8.6). To investigate putative
brain–behavior correlations, we examined the relationship between IQ and FA within the group of subjects
with fragile X with a voxel-based analysis. A few small
clusters, in the superior parts of the prefrontal cortex as
well as the posterior temporal lobe showed a positive
correlation between IQ scores and FA values. These
areas did not correspond to regions of abnormal FA in
the fragile X group as compared to the healthy control
group. No correlation was found between average FA
values obtained in the ROI analysis and IQ scores.
DISCUSSION
In this study, significant differences in FA suggesting alterations in white matter density or coherence,
were detected in females with fragile X syndrome.
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Barnea-Goraly et al.
Fig. 1. A: Voxels that showed significant reduction in white matter fractional anisotropy in fragile X compared to control subjects, mapped onto an
average T1 weighted image of control and fragile X brains. B, C: A three-dimensional representation of the aberrant white matter tracts (shown in yellow)
in relation to the caudate nucleus (shown in red), as drawn from the average image of all subjects, (B) anterior-sagittal view, (C) superior view.
White Matter Tract Alterations in Fragile X Syndrome
85
TABLE I. Clusters in Which Controls had Higher Fractional Anisotropy Than Subjects With Fragile X are Shown With Their Peak
Coordinates in Talairach Space and the Associated Z-Scores, Along With Their Size in Voxels
FA value at the most significant
voxel of difference
Description of extent of cluster
Right sensory-motor tracts
Left caudate extending into the frontal lobe
Left sensory-motor tracts
Talairach coordinates of
most significant voxel (x, y, z)
Cluster size
in voxels
Z-score
Fragile X
group
Control
group
28, 20, 34
18, 23, 3
26, 38, 52
151
106
218
7.51
5.45
5.45
0.38
0.19
0.25
0.465
0.27
0.42
Specifically, the observed differences were seen in
frontal-caudate circuits, as well as in sensory-motor
areas bilaterally. These anisotropy differences suggest
regionally specific alterations of white matter morphology in females with fragile X syndrome.
Aberrant white matter connectivity in sensory-motor
tracts is of potential interest to understanding deficits in
sensory-processing and in sensory-integrative functions that have been observed in fragile X [Baumgardner
et al., 1995; Levitas, 1996; Scharfenaker et al., 1996].
The most commonly reported manifestation of these
deficits is the unusual response of individuals with
fragile X syndrome to sensory stimuli, which is part of
the autistic spectrum behaviors seen in individuals
with this disorder [Reiss and Freund, 1990, 1992;
Baumgardner et al., 1995; Levitas, 1996; Miller et al.,
1999]. Early sensory-processing deficits could adversely affect important domains of development. It has
been postulated that sensory-processing problems impede acquisition of higher cognitive functions, and may
interfere with learning, language development, and
social interactions (summarized in Scharfenaker et al.,
1996). In addition, abnormal reaction to sensory stimulation have been implicated in the development and
maintenance of stereotyped behavior, which is another
common characteristic of individuals with fragile X
syndrome [Baumgardner et al., 1995; Baranek et al.,
1997]. Disruption of white matter tracts leading to brain
regions involved in primary sensory-processing bilaterally, provides a possible anatomical basis for the sensory
dysfunction seen in fragile X syndrome.
The finding of aberrant frontal-caudate pathways
in conjunction with previous observations of enlarged
caudate nucleus in fragile X [Reiss et al., 1995a,b], may
be associated with particular cognitive deficits, and
psychiatric manifestations in fragile X syndrome. The
caudate is part of several circuits that link the basal
ganglia to the frontal cortex. Some of these pathways are
known to play an important role in behavior and cognition [Taylor et al., 1990; Cummings, 1993; Cote and
Crutcher, 2000]. Reports describing the effect of lesions
within these frontal-subcortical circuits suggest that
disturbances in executive function, motor programming, regulation of affect, impulse control, and flexibility in response to environmental cues can occur when
these circuits are damaged [Cummings, 1993; Masterman and Cummings, 1997]. The current findings support the hypotheses put forth in previous studies of
fragile X syndrome that suggest attention dysfunction,
hyperactivity, executive function deficits, and impulse
control problems may be related to frontal-caudate circuit abnormalities [Reiss and Denckla, 1996; Hjalgrim
et al., 1999]. It is difficult to determine which circuits
correspond to the areas where white matter differences
were observed in our study. Future studies using fibertracking techniques will be helpful in determining the
end cortical areas to which these fibers are connected
and will provide more information about the potential
circuits involved in fragile X.
The current observation of alterations in FA in fragile
X syndrome suggests that low levels of FMRP may
contribute to morphological changes in white matter
tracts, possibly due to an influence on neuronal growth
and targeting. FMRP is present in neuronal cell bodies
throughout development [Feng et al., 1997; Agulhon
et al., 1999]. However, it is minimally detected in axons
and not detected in glia, the components of the nervous
system traditionally implicated in axonal growth and
targeting [Hidalgo et al., 1995; Kalil et al., 2000; Sanes
and Thomas, 2000]. Nevertheless, studies suggest an
important role for the target itself in the axonal growth
process. Research in animal models demonstrates that
target cells from the central nervous system are important for the survival, axonal guidance, and synaptic
differentiation of developing neurons [Purves, 1980;
Hsiang et al., 1988]. In addition, once a connection is
made, appropriate neuronal activity is essential for the
correct formation of brain circuits [Katz and Shatz,
1996]. Deleterious effects of suboptimal levels of FMRP
on the structure and function of synapses and dendrites,
the targets for synaptic formation, may help explain the
atypical white matter development in fragile X syndrome observed in the current study.
An increasing body of literature investigating the role
of FMRP in the brain has suggested its importance in
synapse and dendrite formation. Molecular studies demonstrate high levels of FMRP in dendrites, [Devys
et al., 1993; Feng et al., 1997] as well as in synapses
[Feng et al., 1997; Weiler and Greenough, 1999]. Further evidence for the role of FMRP in neuronal development was recently reported by Darnell et al. [2001],
who observed that FMRP–target RNAs encode proteins
with roles in maintaining proper synaptic function and
mediating dendritic and neuronal development. Lack of
FMRP results in immature, irregularly thin, long dendritic spines in post-mortem examination of fragile X
brains as well as in transgenic FMR1 knockout
mice [Rudelli et al., 1985; Hinton et al., 1991; Comery
et al., 1997; Irwin et al., 2001]. Braun and Segal [2000],
reported the first evidence for the importance of
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Barnea-Goraly et al.
FMRP in synaptic function; electrophysiological examination of hippocampal cells from FMR1 knockout mice
demonstrated that they were slower in establishing
synaptic connections, and produced smaller excitatory
synaptic currents than wild-type controls. Taken together, these data suggest the involvement of FMRP in
dendritic spine and synaptic development and function.
Although preliminary, our results suggest that disrupted synaptogenesis, whether due to a putative direct
effect of FMRP on synapse maturation or to an effect on
dendrite development, could disrupt correct interneuronal targeting, synaptic activity, or both. This, in
Fig. 2. Boxplots show FA values from peak locations of significant clusters described in Table I (A, C, D). In right frontal-caudate tracts (B), and in a
control area set in the optic radiations (E, F).
White Matter Tract Alterations in Fragile X Syndrome
87
Fig. 2. (Continued)
turn could result in disrupted white matter density or
coherence.
A clear limitation of the present study is the relatively small sample size. Further research is needed
with larger numbers of subjects and, in particular, males
with fragile X syndrome to confirm these preliminary
results. These studies are ongoing in our laboratory.
Another concern relates to previous findings of enlarged
caudate and ventricular volumes in fragile X [Reiss et al.,
1991, 1995a,b]. This differential morphology of ventricles and caudate nuclei might positionally shift white
matter tracts in the brains of individuals with fragile X.
Therefore, voxels surrounding the ventricles and the
caudate nuclei in controls may contain white matter,
while in subjects with fragile X the same voxels may
contain gray matter, which has lower FA. These spurious FA differences might contribute to the difference in
FA adjacent to the caudate nuclei, but not the FA
differences seen extending from the caudate nuclei to
the frontal lobes.
Finally, a possible relationship between cognition
and white matter anisotropy remains to be clarified.
Our sample groups significantly differed in IQ scores, a
finding that could potentially be associated with differences in white matter structure. However, although the
females with fragile X in this study had IQ scores that
spanned a broad range of abilities—from borderline IQ
through the normal range, no overlap was observed
between brain areas in which FA correlated with IQ and
regions that showed significant between-group differences. This finding suggests that the observed white
matter changes in fragile X are not directly correlated
with cognitive ability. Future studies will need to examine white matter structure in individuals with fragile
X syndrome and a control group with comparable IQ
scores to further investigate this issue.
In conclusion, this study shows preliminary evidence
of white matter tract abnormalities in females with
fragile X syndrome. These findings may help to provide
a better understanding of the neuroanatomical and,
potentially, the functional impact of decreased FMRP
levels in the brain of persons with fragile X.
ACKNOWLEDGMENTS
The research presented in this manuscript has been
supported by the following grants to Dr. Allan L. Reiss:
NIH Grants MH01142, HD31715, MH50047, the Packard Foundation, the Sinclair Fund, and the Lynda and
Scott Canel Fund for Fragile X Research. The author
thanks Ben Krasnow, Eric Schmitt, and Leanne Tamm
for their help with image processing and manuscript
preparation.
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